Our goal is to discover ligands that bind to and stabilize the soluble isoform of the human prion protein (HuPrPc). Some of these ligands may also inhibit the conversion of HuPrPSc into the oligomeric, diseasecausing isoform (HuPrPSc). There are few or no well-validated molecules that bind to PrPc and their absence is a key gap in our ability to probe the function of PrPc and attenuate the pathologies associated with PrPSc. Using a virtual screening approach, large libraries of organic molecules will be docked against the structure of PrPc. High-scoring compounds will be tested for binding biophysically, controlling for non-specific inhibition to which anti-amyloid inhibitors are prone. Our long term goal is to use specific PrP ligands to investigate the protein's biological function and as potential leads for drug discovery; our immediate intent is to discover and fully characterize them. The specific aims are: Aim 1. Structure-based virtual screens to discover ligands that stabilize PrPc. We will dock a set of 100,000 fragment-like and one Million lead-like molecules against the structure of PrPc; all are commercially available so the predictions may be rapidly tested. Each compound is fit into one of two clefts in PrPc and ranked by complementarity to the protein. Additionally, we will predict the targets of known reagents active in cell culture against prion aggregation, using a second computational approach. Aim 2. To test high-scoring docking hits for efficacy in direct binding assays. Docking hits will be tested in a recently developed thermal denaturation upshift assay. We will also develop new assays that measure binding using isothermal titration calorimetry and surface plasmon resonance. Such assays will be critical to understanding mechanism of the ligands and, in the longer term, improving their affinities. Aim 3. To control for specific inhibition and investigate the role of colloidal aggregates on prions. Many putatively specific inhibitors of amyloid act not by direct-binding, classical mechanisms, but rather as promiscuous, colloid-based inhibitors. These colloids sequester the protein monomers, preventing amyloid formation. We extend initial studies of this effect to other well-known anti-amyloid inhibitors, and also control for it as a mechanism in the molecules that we ourselves discover and test in Aims 1 and 2.